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So in this final step, an ATP is used to phosphorylate a UDP and that regenerates that UTP. | Overview of Gluconeogenesis .txt |
So once again, let's very briefly overview this entire process. | Overview of Gluconeogenesis .txt |
So we ingest a meal that is rich in sugar molecules. | Overview of Gluconeogenesis .txt |
The blood level of glucose basically increases. | Overview of Gluconeogenesis .txt |
Our liver wants to maintain that and decrease it back to normal. | Overview of Gluconeogenesis .txt |
So the liver cells uptake the glucose molecules to trap the glucose within the cell and to prevent it from leaving the cell, they use hexacionase and an ATP to form glucose six phosphate. | Overview of Gluconeogenesis .txt |
Next, the glucose six phosphate is transformed into glucose one phosphate by the activity of phosphor glucom Utase. | Overview of Gluconeogenesis .txt |
Now, once we form the glucose one phosphate, we want to activate the molecule, make it much more reactive and increase its energy. | Overview of Gluconeogenesis .txt |
And so what we do is we use UDP glucose pyrophosphorylase to basically transfer a group from UTP onto the glucose one phosphate and that kicks off a Pyrophosphate, as shown in this diagram. | Overview of Gluconeogenesis .txt |
So we generate the UDP glucose and the Pyrophosphate. | Overview of Gluconeogenesis .txt |
Now this reaction isn't really product favored. | Overview of Gluconeogenesis .txt |
And to make it product favored to drive the formation of UDP glucose, the Pyrophosphate is cleaved by water into two orthophosphate molecules. | Overview of Gluconeogenesis .txt |
So Pyrophosphate is hydrolyzed into two orthophosphates and this drives the formation of UDP glucose. | Overview of Gluconeogenesis .txt |
Now, once we have a bunch of these UDP glucose molecules, the enzyme we call glycogenin, not shown in this diagram, basically uses the UDP glucose molecules to generate a primer, a short sequence of glucose molecules that are linked by alpha one four glycocitic bonds. | Overview of Gluconeogenesis .txt |
Because glycogen synthase needs that primer to begin the elongation process, to begin extending that glycogen. | Overview of Gluconeogenesis .txt |
So now we take the UDP glucose. | Overview of Gluconeogenesis .txt |
In the presence of the primer, the enzyme glycogen synthase basically increases or attaches this glucose onto that primer. | Overview of Gluconeogenesis .txt |
In the process, we form a UDP molecule. | Overview of Gluconeogenesis .txt |
Now, once we continually add these glucose molecules, eventually we want to actually branch that glycogen. | Overview of Gluconeogenesis .txt |
And so that's where step six takes place. | Overview of Gluconeogenesis .txt |
Glycogen branching enzyme is responsible for creating those alpha one six linkages which lead to the branching points. | Overview of Gluconeogenesis .txt |
And for this process to actually continually taking place, we have to take the UDP that we form here and regenerate back that UTP. | Overview of Gluconeogenesis .txt |
And that's where step seven comes into play. | Overview of Gluconeogenesis .txt |
The enzyme nucleotide di nucleoside diphosphokinase uses an ATP to attach it onto the UDP here, and that forms that UTP. | Overview of Gluconeogenesis .txt |
And so once we reject generate the supply of UTP, this process can continue taking place. | Overview of Gluconeogenesis .txt |
And so as long as we're forming the UTP glucose molecules, we can continually build that glycogen and extend that glycogen polymer. | Overview of Gluconeogenesis .txt |
So once we amplify a gene of interest, once we produce many identical copies of a single gene, what exactly do we do next? | Restriction Map and Gel Electrophoresis.txt |
Well, usually the next process is to take that gene and produce a restriction map for that particular gene. | Restriction Map and Gel Electrophoresis.txt |
Now what exactly is a restriction map? | Restriction Map and Gel Electrophoresis.txt |
Well, let's suppose that we have the following ruler and this ruler describes as our gene of interest that we amplify. | Restriction Map and Gel Electrophoresis.txt |
So this is the double stranded DNA molecule that describes the interest, that describes the gene that we're studying. | Restriction Map and Gel Electrophoresis.txt |
So what a restriction map is? | Restriction Map and Gel Electrophoresis.txt |
It's basically a description of all the different locations found on this gene where our restriction enzymes can bind to and cleave that gene. | Restriction Map and Gel Electrophoresis.txt |
So for example, let's suppose this gene has three different restriction sites. | Restriction Map and Gel Electrophoresis.txt |
So here, here and here. | Restriction Map and Gel Electrophoresis.txt |
And what that means is a type of restriction enzyme can bind onto these three locations and cleave that particular gene and those locations. | Restriction Map and Gel Electrophoresis.txt |
So that's what we mean by a restriction map. | Restriction Map and Gel Electrophoresis.txt |
Now what exactly is the procedure in creating the restriction map? | Restriction Map and Gel Electrophoresis.txt |
Well, the restriction map basically involves or creating the restriction map involves a process known as gel electrophoresis. | Restriction Map and Gel Electrophoresis.txt |
And this will be the focus of this lecture. | Restriction Map and Gel Electrophoresis.txt |
So we're going to focus on the process of gel electrophoresis that is used in the creation of the restriction map for any particular gene. | Restriction Map and Gel Electrophoresis.txt |
So let's suppose we look at diagram A. | Restriction Map and Gel Electrophoresis.txt |
In diagram A, we have the following gene shown in black. | Restriction Map and Gel Electrophoresis.txt |
So this is our gene. | Restriction Map and Gel Electrophoresis.txt |
Let's suppose we expose this gene, this gene here, to a specific type of restriction enzyme we're going to call enzyme number one. | Restriction Map and Gel Electrophoresis.txt |
Now what happens is once we expose the gene to this restriction enzyme, the restriction enzyme cuts or cleaves this gene at two different locations. | Restriction Map and Gel Electrophoresis.txt |
So at this location, somewhere here, and in this location somewhere here. | Restriction Map and Gel Electrophoresis.txt |
So at the end, once we expose this gene to this enzyme, we get three different DNA fragments. | Restriction Map and Gel Electrophoresis.txt |
So we have DNA fragment one, DNA fragment two, and DNA fragment three. | Restriction Map and Gel Electrophoresis.txt |
And these fragments all came from this entire gene. | Restriction Map and Gel Electrophoresis.txt |
Now once we obtain these fragments, we now expose the fragments to the process of gel electrophoresis. | Restriction Map and Gel Electrophoresis.txt |
And what this process does is it ultimately separates these three DNA fragments that came from the gene based on their physical size. | Restriction Map and Gel Electrophoresis.txt |
So what exactly is gelatrophresis? | Restriction Map and Gel Electrophoresis.txt |
Well, it's basically the process by which we take our fragments. | Restriction Map and Gel Electrophoresis.txt |
We place them into a special type of porous gel and then we allow those fragments to move through the porous of the gel as a result of an electric field that exists within that gel. | Restriction Map and Gel Electrophoresis.txt |
So we take the apparatus, we connect the apparatus to a voltage source and that creates an electric potential difference, a voltage difference between the two sides of that electrophoresis setup. | Restriction Map and Gel Electrophoresis.txt |
And so what happens is, because we connected to our battery source, one end of that plate will have a negative charge. | Restriction Map and Gel Electrophoresis.txt |
So that will be the count node and the other end will have a positive charge that will be the anode. | Restriction Map and Gel Electrophoresis.txt |
Now remember, DNA contains a negative charge as a result of all those phosphate groups. | Restriction Map and Gel Electrophoresis.txt |
And so all these DNA fragments that came from the gene will contain negative charge and they will move from the calcio, the negatively charged side, to the anode, the positively charged side. | Restriction Map and Gel Electrophoresis.txt |
So all these fragments will move along the same direction, but they will move at different speeds. | Restriction Map and Gel Electrophoresis.txt |
And that's because if we examine the gel inside this setup, that gel is basically a special type of polymer that contains many different pores. | Restriction Map and Gel Electrophoresis.txt |
And these pores basically contain a certain size to them. | Restriction Map and Gel Electrophoresis.txt |
And so the larger DNA fragments, the larger molecules will find it more difficult to move along these pores, while the smaller fragments will find it easier to move along and through these pores because of their smaller physical size and smaller physical weight. | Restriction Map and Gel Electrophoresis.txt |
So in gel electrophoresis, different fragments are separated on the basis of physical size. | Restriction Map and Gel Electrophoresis.txt |
The larger molecules are not able to move as quickly as the smaller ones through the pores of that gel. | Restriction Map and Gel Electrophoresis.txt |
Now, since DNA fragments are all negatively charged, they all move along the same direction. | Restriction Map and Gel Electrophoresis.txt |
They always move from the cathode, the negatively charged side, to the anode, the positively charged side. | Restriction Map and Gel Electrophoresis.txt |
And this voltage difference is created because this entire structure is connected to a battery source. | Restriction Map and Gel Electrophoresis.txt |
So this is what gel electrophoresis is. | Restriction Map and Gel Electrophoresis.txt |
So the way that we create the restriction map is by exposing this initial gene to many different types of restriction enzymes. | Restriction Map and Gel Electrophoresis.txt |
For example, in case A, we expose the gene to restriction enzyme number one. | Restriction Map and Gel Electrophoresis.txt |
And we form three different fragments, as shown, that have these different sizes. | Restriction Map and Gel Electrophoresis.txt |
Now, in case B, we take that same initial gene, but now we expose it to a different restriction enzyme which cuts at different locations along that gene. | Restriction Map and Gel Electrophoresis.txt |
So now, instead of producing these three fragments, we only produce two fragments because this gene only contains one side, one location where this restriction enzyme number two can actually act on. | Restriction Map and Gel Electrophoresis.txt |
So now we produce fragment four and fragment five. | Restriction Map and Gel Electrophoresis.txt |
And once again, we expose these two fragments. | Restriction Map and Gel Electrophoresis.txt |
We place these two fragments into our gel and now we have the separation based on size. | Restriction Map and Gel Electrophoresis.txt |
And we can compare this diagram to this diagram and we can use the information obtained to basically create a restriction map. | Restriction Map and Gel Electrophoresis.txt |
So if we examine the following diagram right over here, we see that for this particular case, this fragment is this fragment here. | Restriction Map and Gel Electrophoresis.txt |
And notice it is closest to the anode because it is the smallest. | Restriction Map and Gel Electrophoresis.txt |
And it is able to move the farthest along and through the porous of the gel. | Restriction Map and Gel Electrophoresis.txt |
This fragment is basically fragment two. | Restriction Map and Gel Electrophoresis.txt |
This fragment is fragment three. | Restriction Map and Gel Electrophoresis.txt |
Now, what about this case? | Restriction Map and Gel Electrophoresis.txt |
Well, notice that fragment three is almost the same size as fragment four. | Restriction Map and Gel Electrophoresis.txt |
And so these two will correspond to the same exact position, horizontal position, along the following diagram. | Restriction Map and Gel Electrophoresis.txt |
Now, this is the largest fragment of these five fragments. | Restriction Map and Gel Electrophoresis.txt |
And so it will be found highest farthest up along the following plate. | Restriction Map and Gel Electrophoresis.txt |
So we have the largest fragment. | Restriction Map and Gel Electrophoresis.txt |
These two fragments are about the same size. | Restriction Map and Gel Electrophoresis.txt |
Then the next fragment and the smallest fragment, fragment number one. | Restriction Map and Gel Electrophoresis.txt |
So let's take a marker and let's label these just so we know. | Restriction Map and Gel Electrophoresis.txt |
So this is fragment number one, fragment number two, fragment number three, fragment number four and fragment number five. | Restriction Map and Gel Electrophoresis.txt |
So this is what gel electrophoresis is. | Restriction Map and Gel Electrophoresis.txt |
Now, when we're, we'll get into biochemistry, we're going to see that we can use gel electrophoresis not only for DNA molecules, but we can also use for proteins. | Restriction Map and Gel Electrophoresis.txt |
We can basically separate our proteins by their physical size using the same exact process of gel electrophoresis. | Restriction Map and Gel Electrophoresis.txt |
Previously we discussed restriction enzymes and we said we can use these restriction enzymes to cut our DNA molecule into smaller fragments known as restriction fragments. | Southern and Northern Blotting.txt |
Now, once we have these DNA restriction fragments we can basically study these fragments, we can analyze them, we can manipulate them, we can amplify them, make many copies and we can do all sorts of different things with these DNA fragments. | Southern and Northern Blotting.txt |
Now let's suppose we take the DNA molecule, we expose it to our restriction enzymes and we create these many DNA fragments. | Southern and Northern Blotting.txt |